Efficacy of luteal estrogen administration and an early follicular Gonadotropin-releasing hormone antagonist priming protocol in poor responders undergoing in vitro fertilization

OBJECTIVES: We investigated whether luteal estrogen administration and an early follicular Gonadotropin-releasing hormone antagonist (E/G-ant) priming protocol improves clinical outcomes in poor responders to controlled ovarian stimulation for in vitro fertilization (IVF)-embryo transfer, and identified underlying mechanisms. METHODS: This restrospective study consisted of 65 poor responders who underwent the E/G-ant priming protocol. Sixty-four other poor responders undergoing conventional protocols without pretreatment were included as the control group. Clinical outcomes were compared between 2 groups. RESULTS: The E/G-ant priming protocol group exhibited improvements over the control group in terms of the number of retrieved oocytes (3.58±2.24 vs. 1.70±1.45; P=0.000), mature oocytes (2.68±2.11 vs. 1.65±1.23; P=0.000), fertilized oocytes (2.25±1.74 vs. 1.32±1.26; P=0.001), good embryos (1.62±0.91 vs. 1.14±0.90, P=0.021). Day 3 follicle-stimulating hormone (FSH; 8.40±4.84 vs. 16.39±13.56; P=0.000) and pre-ovulation progesterone levels (0.67 vs. 1.28 ng/mL; P=0.016) were significantly higher in the control group than in the E/G-ant priming group. The overall rate of positive human chorionic gonadotropin tests was higher in the E/G-ant priming group than in the control group (32.3% vs.16.1%; P=0.039). Also, clinical pregnancy rate (26.2% vs. 12.5%; P=0.048) and the rate of live births (23.1% vs. 7.1%; P=0.023) were significantly higher in the E/G-ant priming group than in the control group. CONCLUSION: The E/G-ant priming protocol would lead to promising results in poor responders to IVF by suppressing endogenous FSH and by preventing premature luteinization.

A high response to controlled ovarian stimulation induces premature luteinization with a negative impact on pregnancy outcomes in a gonadotropin-releasing hormone antagonist cycle / 대한생식의학회지

OBJECTIVE: The goal of this study was to investigate the relationship between serum progesterone (P4) levels on the day of human chorionic gonadotropin (hCG) administration and the pregnancy rate among women undergoing controlled ovarian stimulation for in vitro fertilization (IVF) or intracytoplasmic sperm injection-embryo transfer (ICSI-ET) using a flexible antagonist protocol. METHODS: This prospective study included 200 IVF and ICSI-ET cycles in which a flexible antagonist protocol was used. The patients were divided into five distinct groups according to their serum P4 levels at the time of hCG administration (0.80, 0.85, 0.90, 0.95, and 1.00 ng/mL). The clinical pregnancy rate (CPR) was calculated for each P4 interval. Statistically significant differences were observed at a serum P4 level of 0.9 ng/mL. These data suggest that a serum P4 concentration of 0.9 ng/mL may represent the optimal threshold level for defining premature luteinization (PL) based on the presence of a significant negative impact on the CPR. RESULTS: The CPR for each round of ET was significantly lower in the PL group defined using this threshold (25.8% vs. 41.8%; p=0.019), and the number of oocytes retrieved was significantly higher than in the non-PL group (17.3+/-7.2 vs. 11.0+/-7.2; p=0.001). Elevated serum P4 levels on the day of hCG administration were associated with a reduced CPR, despite the retrieval of many oocytes. CONCLUSION: Measuring serum P4 values at the time of hCG administration is necessary in order to determine the optimal strategy for embryo transfer.

Molecular mechanisms of luteinization

Dynamic changes in steroidogenesis occur in ovarian granulosa cells during ovulation after the LH surge. The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells undergoing luteinization during ovulation. These rapid changes in StAR and Cyp19a1 gene expression after the LH surge efficiently facilitate progesterone production, which plays a crucial role in ovulation and the following luteinization. Recently, it has become clear that epigenetic regulation such as histone modifications and DNA methylation play a key role in gene expression through the chromatin remodeling of the promoter region. This study reports the in vivo evidence that epigenetic mechanisms including histone modifications, DNA methylation and chromatin remodeling are involved in the rapid changes of StAR and Cyp19a1 gene expression in granulosa cells undergoing luteinization during ovulation.

Cultura de células da granulosa humanas com fenótipo de fase folicular: influência do sistema quimicamente definido na morfologia, ultraestrutura, secreção de esteroides e relaxina / Culture of human granulosa cells with follicular phase phenotype:influence of chemically defined system on morphology, ultrastructure,secretion of steroids and relaxin

Está bem descrito na literatura o padrão de cultivo de células da granulosa (CG) humanas que perpetua a luteinização, simulando a fase lútea do ciclo. Nesse sistema, há redução na secreção de estradiol (E2) e aumento na síntese de progesterona (P4) e relaxina (RLN). Objetivamos padronizar um sistema de cultura livre de soro, com o intuito de reverter o processo de luteinização de CG obtidas em ciclos de fertilização in vitro (FIV), pré-luteinizadas pela gonadotrofina coriônica humana (hCG), para aplicação na maturação in vitro de folículos ovarianos pré-antrais. Foi feito estudo experimental com GC obtidas de 10 mulheres em tratamento de reprodução assistida. As CG foram cultivadas em α-MEM contendo IGF-I, ITS, androstenediona, PVP-40 (meio quimicamente definido) ou TCM-199 contendo FSH/soro. Após 48, 96 e 144 horas, foram avaliados: morfologia das culturas, produção de E2, P4 (Quimioluminescência/Immulite), RLN (Elisa) e ultraestrutura (Microscopia Eletrônica). Os dados foram analisados por Anova e regressão linear com efeitos mistos (SAS versão 9.0). Células cultivadas em α-MEM apresentam alta capacidade estrogênica e padrão de produção hormonal característico da fase folicular, mantendo características morfológicas/ultraestruturais semelhantes a células in vivo. No sistema de cultura padronizado, as CG não completam in vitro o processo de luteinização deflagrado pela hCG, assumindo fenótipo de fase folicular.

It is well described in the literature the granulosa cells (GC) culture pattern that perpetuates human luteinizing simulating the luteal phase of the cycle. In this system, there is a reduction in the secretion of estradiol (E2) and increased synthesis of progesterone (P4) and relaxin (RLN). We aim to standardize a serum-free culture system, in order to reverse the luteinization process of GC obtained in IVF cycles, pre-luteinized by hCG, for use in in vitro maturation of preantral ovarian follicles. An experimental study was conducted with GC obtained from10 women undergoing treatment for assisted reproduction. The GC were cultured in α-MEM containing IGF-I, STI, androstenedione, PVP-40 (chemically defined medium) or TCM-199 containing FSH/serum. After 48, 96 and 144 h were analyzed: culture morphology, concentrations of E2, P4 (Chemioluminescence/Immulite), and RLN (Elisa), and ultrastructure (ElectronMicroscopy). Data were analyzed by Anova and linear mixed-effects regression (SAS version9.0). Cells cultured in α-MEM present estrogenic capacity and pattern of hormone production characteristic of the follicular phase, maintaining morphological/ultrastructural features similar that in vivo cell. In standard culture system, the CG not completes in vitro luteinization process triggered by hCG, assuming follicular phase phenotype.

Cessation of gonadotropin-releasing hormone antagonist on triggering day in flexible multiple-dose protocol: A randomized controlled study / 대한생식의학회지

OBJECTIVE: To investigate outcomes of stimulated IVF cycles in which GnRH antagonist was omitted on the ovulation triggering day. METHODS: A total of 86 women who underwent controlled ovarian hyperstimulation with recombinant FSH and GnRH antagonist flexible multiple-dose protocols were recruited and prospectively randomized into the conventional group (group A) or cessation group (group B). The GnRH antagonist, 0.25 mg/day of cetrorelix, was started when the leading follicle reached 14 mm in diameter and was continuously administered until the hCG triggering day (group A, 43 cycles) or until the day before hCG administration (group B, 43 cycles). The maturity of oocytes, fertilization rate, embryo quality, and implantation and clinical pregnancy rates were evaluated. RESULTS: The duration of ovarian stimulation, total dose of gonadotropins, serum estradiol levels on hCG administration day, and number of oocytes retrieved were not significantly different between the two groups. The total dose of GnRH antagonist was significantly lower in group B than group A (2.5+/-0.9 vs. 3.2+/-0.8 ampoules, p<0.05). There was no premature luteinization in any of the subjects. The proportion of mature oocytes and fertilization rate were not significantly different in group B than group A (70.7% vs. 66.7%; 71.1% vs. 66.4%, respectively). There were no significant differences in the implantation or clinical pregnancy rates. CONCLUSION: Our prospective randomized study suggested that cessation of GnRH antagonist on the hCG administration day during a flexible multiple-dose protocol could reduce the total dose of GnRH antagonist without compromising its effects on pregnancy rates.

Ovarian hemangioma with stromal luteinization and HCG-producing mononucleate and multinucleate cells of uncertain histogenesis: A rare co-existence with therapeutic dilemma.

A 21 year old female presented with amenorrhea, hirsutism and change in voice along with an elevated serum β-HCG (human chorionic gonadotrophin) level and normal CA-125 level. Laparotomy revealed an enlarged right ovary measuring 6 × 5 × 1 cms with presence of an ovarian hemangioma along with stromal luteinization and HCG producing mononucleate as well as multinucleate cells of uncertain histogenesis on histopathological examination. Immunohistochemistry for inhibin and calretinin were positive in the luteinized component whereas β-HCG and Ki-67 were positive in the multinucleate cell component. The diagnostic rarity and therapeutic dilemma of such a rare mixed tumor within a single ovary has proven to be an exceptional case and an excellent investigative opportunity.

Progesterone/estradiol ratio in the late follicular phase of long gonadotropin-releasing hormone agonist cycles did not differ between conceived and not-conceived women

There is a challenging debate on the effect of premature luteinization on the clinical outcome of 'controlled ovarian hyperstimulation' [COH] using long 'gonadotropin-releasing hormone agonist' [GnRHa] cycles. Premature luteinization is defined as late follicular progesterone/estradiol ratio more than 1 on the day of human chorionic gonadotropin [HCG] administration. We carried out a retrospective case-control study on 75 conceived cases versus 75 not-conceived control women, receiving long GnRHa cycles in their first cycle of treatment. Premature luteinization developed in 15% of the case group vs. 22% of the control group. Neither the late follicular progesterone/estradiol [P/E2] ratio was significantly different between the two groups, nor the day 3 follicle stimulating hormone [FSH], serum estradiol level on the HCG day, total amount of human menopausal gonadotropins ampoules, number of follicles, retrieved oocytes and transferred embryos. Endometrial thickness was significantly more in the pregnant women than in the non-pregnant group. Premature luteinization seems not to adversely affect the clinical outcome of COH

The expression of p57(kip2) in normal ovaries and ovarian tumors / 대한산부인과학회지

OBJECTIVES: The purpose of this study is to investigate the expression of CDK (Cyclin dependent kinase) inhibitor, p57(kip2) in human ovarian corpus luteum, benign and malignant ovarian tumors. METHODS: 46 women undergoing laparoscopic surgery or laparotomy for ovarian tumors were enrolled. Total 46 formalin-fixed, paraffin-embedded sections of corpus luteum, benign and malignant ovarian tumors were stained by immunohistochemistry for expression of p57(kip2). RESULTS: p57(kip2) was stained in theca cell of growing follicle but not induced in human corpus luteum. There was the expression of p57(kip2) in mature teratoma, immature teratoma and endometrioma but not in epithelial ovarian tumors. CONCLUSIONS: These results showed that p57(kip2) expression may be not important in luteinization of the ovary and seemed not to play a role in development of epithelial ovarian tumors. However, it may involve pathogenesis of mature teratoma, immature teratoma and endometrioma.

Do all regularly menstruating women ovulate in each cycle?

Women may experience anovular menstruation due to some pathophysiological causes which can be detected either by invasive histological examination as well as noninvasive serial ultrasound test. The women who are regularly menstruating without ovulation in each cycle were identified in this study. In a tertiary level infertility care centre of Bangladesh in Dhaka infertile population was the subject of the study. The serial ultrasound noninvasive procedure is used for diagnosis of anovular menstruation and found very much helpful.

GnRH Antagonist Multiple Dose Protocol in Controlled Ovarian Hyperstimulation for In Vitro Fertilization and Embryo Transfer Compared with GnRH Agonist Long Protocol / 대한산부인과학회잡지

OBJECTIVE: To evaluate the efficacy of GnRH antagonist multiple dose protocol (MDP) in controlled ovarian hyperstimulation (COH) for in vitro fertilization and embryo transfer (IVF-ET) comparing with the standard GnRH agonist long protocol (GnRH-a LP). METHODS: From January 2000 to September 2002, 57 infertile women with tubal factor alone who had undergone IVF-ET were enrolled in the present study. Study group consisted of 28 patients in 28 cycles in which GnRH antagonist Cetrorelix 0.25 mg was given daily when the leading follicle reached 14 mm in mean diameter until the human chorionic gonadotropin (hCG) injection. Control group consisted of 29 patients in 29 cycles in which COH was performed using standard GnRH-a luteal LP. RESULTS: Patient's characteristics were comparable in both groups. Premature luteinization was not developed in all patients in each group. The number of ampules and duration of exogenous gonadotropins required were significantly lower in the study group than those in the control group (por=14 mm diameter on the day of hCG injection, the number of oocytes retrieved, fertilization rate, and the number of grade I, II embryos between the two groups, but the numbers of mature oocytes retrieved and fertilized oocytes were significantly lower in the study group than in the control group (p<0.01, p<0.01). The clinical pregnancy rate seemed to be lower in the study group, but the difference did not achieve significance (28.6% vs 34.5%). There were also no differences in the miscarriage rate and multiple pregnancy rate between the two groups. CONCLUSION: This study demonstrates that GnRH antagonist Cetrorelix MDP can result in the comparable pregnancy outcome as the GnRH-a LP and furthermore reduce the total dose of gonadotropins and duration of stimulation.

A Case of Ruptured Juvenile Granulosa Cell Tumor / 대한산부인과학회잡지

Sex cord-stromal tumors of the ovary are the third most common types of neoplasms that develop in the ovary and account for about 5-8% of all ovarian malignancies. Juvenile granulosa cell tumor (JGCT) is one of the sex cord-stromal tumors of the ovary has distinct differences from adult granulosa cell tumor (AJCT) with regard to clinical and pathological features as well as biological behavior most frequently occuring in the first two decades of life. Usually Call-exner bodies are rare, and luteinization is frequent in JGCT. The tumor may be solid, cystic, or both. In premenarcheal girls, juvenile granulosa cell tumor usually (82%) elicits the signs of sexual precocity. The tumor should removed as soon as the diagnosis is established. Surgery is the best treatment choice for low stage juvenile granulosa cell tumor in children, but for those with high stage juvenile granulosa cell tumor or recurrent tumor, the best treatment and sensitivity of tumor to radiation therapy and chemotherapy have not yet been determined clearly. About 90% are diagnosed in early stage so, prognosis of juvenile granulosa cell tumor in children is good in most cases, but tumor with more advanced stage has worse clinical outcome correlated with its stage, presence of ruptures, grade of nuclear atypia, degree of mitotic activity. And the clinical stage at the time of diagnosis is considered most important prognostic factor. We experienced a case of ruptured juvenile granulosa cell tumor so, we present a case with brief review of literature.

Effects of Nitric Oxide on the Maturation of Mouse Oocyte in vitro / 대한산부인과학회잡지

OBJECTIVE: Nitric oxide (NO) produced in ovary may contribute to follicle maturation, ovulation, oocyte maturation and luteinization. In this study, the effect of nitric oxide on the spontaneous maturation of mouse oocyte was observed. Method: The index of oocyte maturation was checked by the germinal vesicle breakdown (GVBD) and appearance of polar body (PB) under microscope in the denuded oocytes and oocyte-cumulus complexes (OCCs) from mouse ovarian follicles after 24 hours pregnant-mare serum gonadotropin treatment. RESULTS: The GVBD appeared 50 %, 1 hour and 80 %, 2 hrs after changes of oocytes from dibutyryl cAMP (dbcAMP, 0.5 mM) contained media into dbcAMP-free media. dbcAMP (0.5 mM) completely blocked the GVBD until 24 hrs but dbcGMP (5 mM) delayed the GVBD by 1 hr. Sodium nitroprusside, the NO generator, inhibited the GVBD dose-dependently at 2 hr incubation in denuded and OCCs. The appearance of GVBD was not different between control and dbcGMP or SNP in denuded oocytes and OCCs at 24 hrs incubation. The guanylate cyclase activity in denuded oocyte cytosol was not detected whereas the guanylate cyclase activity in OCCs cytosol was 1.3 nmole/min/mg protein which was increased about 3 times by SNP (100 micrometer). CONCLUSION: These results suggest that the NO in ovary may delay the spontaneous oocyte maturation in early stage by acting on the maturation signaling protein as well as guanylate cyclase.

A Case of Juvenile Granulosa Cell Tumor of the Ovary

Juvenile granulosa cell tumor (JGCT) is one of the sex cord stromal tumors of the ovary ocurring in the first two decades of life. These tumors are different from adult granulosa cell tumor (AJCT) with regard to clinical and pathological fetures. Follicles are often irregular, Call-exner bodies are rare, and luteinization is frequent. The tumor may be solid, cystic, or both. The most common presenting symptoms are abnormal uterine bleeding and pain. Breast swelling, pain and tenderness may also be associated with unopposed estrogen secretion by granulosa cell tumors. The tumor should be removed as soon as the diagnosis is estabilished. The juvenile granulosa cell tumor has a good overall prognosis because fewer than 5% of these tumors in children are malignant.